Cell differentiation along the crypt-to-villus axis in the intestinal tract is a dynamic and programmed process, however, the regulating molecular mechanisms remain largely elusive. The intestinal environment is complex and heterogeneous, thus, cell culture lines that could be used as models for intestinal epithelium should allow for the study of differentiation in a defined and controlled setting. Clonal derivatives of the HT29 human adenocarcinoma cell line can be induced to differentiate in vitro thereby, acquiring both morphologic and biochemical characteristics of absorptive and secretory cells. The objective of this Physician Scientist Award is to provide training and experience in the study of intestinal epithelial differentiation based on the hypothesis that this differentiation is the result of gene regulation by a group of cis- and trans-acting regulatory elements, many of which may be intestinal specific. Along with didactic' and laboratory training, Phase I will build upon preliminary results already obtained. Using the technique of subtraction cloning, a novel cDNA (A4) has been isolated whose expression is transcriptionally activated with differentiation of the HT29-l8 and HT29-l8-C1 clones. The A4 gene displays both horizontal and vertical gradients of expression in the human intestine. These results indicate that A4 is a suitable reagent to study the mechanisms involved in activating differentiation-dependent genes in this cell culture system. Initial analysis of the A4 putative promoter has already revealed a potential positive cis-acting element. During Phase I, particular attention will be focused on learning the molecular biology techniques needed to identify and analyze the A4 promoter region and its regulatory elements. The protein encoded by A4 will also be characterized and the developmental expression of the A4 gene explored. Phase II will be an intensive research period to obtain further laboratory experience and to gather preliminary data needed to pursue an independent research career. This stage will deal with identifying other differentially expressed genes and characterizing their regulation in order to search for a unifying or collective pattern of regulation in this cell model of intestinal epithelial differentiation that may then be applied to an in vivo system.